This invention relates generally to the generation of aptamers and to the use of aptamers as diagnostic and therapeutic agents. More particularly, the present invention relates to a method of using combinatorial chemistry to prepare novel oligonucleotide sequences having at least one thiophosphate replacement in the phosphate backbone and that have enhanced target binding properties.
Without limiting the scope of the invention, its background is described in connection with oligonucleotide agents and with methods for the isolation and generation thereof.
Oligonucleotide agents have been shown to have functional activity in vitro and thus the promise of therapeutic potential. Some of these agents are believed to operate via mechanisms such as the sequence-specific antisense translation arrest of mRNA expression or through direct binding to protein targets where they function as xe2x80x9cdecoysxe2x80x9d. While oligonucleotide agents show therapeutic promise, various pharmacological problems must first be overcome.
Oligonucleotide agents have been used as high specificity therapeutic agents in vitro. High sensitivity to nuclease digestion, however, makes oligonucleotide agents unstable and thus impracticable for in vivo administration by either intravenous or oral routes.
From the foregoing it is apparent the there is a need in the art for methods for generating high binding, nuclease resistant oligonucleotide that retain their specificity. Also needed are compounds and methods that permit the generation of high binding, high specificity, nuclease resistant oligonucleotide agents that have an improved half-life and are target specific.
Aptamers may be defined as nucleic acid molecules that are selected from random or high-sequence diversity libraries due to their ability to bind with a target. An iterative process known as in vitro selection is used to enrich the library for species with high affinity to the target. The iterative process involves incubation of the library with the target, separation of target bound oligonucleotide (xe2x80x9cTBOxe2x80x9d) sequences from free TBO and amplification of the bound and thus selected TBO population to enrich the library. Amplification may be effected enzymatically, for example, using a thermostable DNA polymerase in a polymerase chain reaction (xe2x80x9cPCRxe2x80x9d). The result is a sub-library populated with a small subset of sequences that have a high affinity for the target. The library is then subcloned to sample and preserve the target specific DNA, RNA or mixed sequences selected. These xe2x80x9clead compoundsxe2x80x9d may then be studied in further detail to elucidate the mechanisms by which they interact with the target.
The present invention is directed to the generation of aptamers that are nuclease resistant and therapeutically effective. In one embodiment of the present invention, a method is provided of selection of modified oligonucleotide aptamers wherein the modification of constituent nucleotides confers nuclease resistance to the oligomer. In a first step, a random single-stranded combinatorial library is chemically synthesized wherein the synthetic library includes at least one set of 5xe2x80x2 and 3xe2x80x2 primer segments flanking a randomized region. Next, the random combinatorial library is amplified and modified using enzymatic synthesis, for example, by PCR. The synthetic reaction is provided with a pool of four nucleotides including dA, dT, dG and dC, in which at least one, for example dA is used. Not all four nucleotides, however, are chemically modified prior to use in the reaction. The modified oligonucleotide library is then placed into contact with the target molecule. Nucleotides that bind the specific target are isolated from those which do not. Next, oligonucleotides binding to the target molecule are again amplified enzymatically in the presence of the desired modified nucleotide substrates. The isolation and amplification steps are repeated iteratively until at least one oligonucleotide population of defined sequence is obtained. Sequencing and cloning of target-selected aptamers is used to isolate, preserve and enhance the conformation of the aptamer.
In one embodiment of the present invention the nucleotide modification conferring nuclease resistance is thiolation of one or both of the non-bridging oxygens around the phosphorus. The present inventors have shown that various backbone modifications, such as the phosphorothioates and phosphorodithioates, may render aptamers more nuclease resistant while still permitting efficient uptake by cells (Wang, S., Lee, R. J., Cauchon, G. G., Gorenstein, D. G. and Low, P. S., Proc. Natl. Acad. Sci., U.S.A. (1995) 92: 3318).
Unfortunately, it has become apparent that oligonucleotides possessing high thiophosphate backbone substitutions appear to be xe2x80x9cstickierxe2x80x9d towards proteins than normal phosphate esters, attributable to non-specific interactions. (Cho, Y. S., Zhu, F. C., Luxon, B. A. and Gorenstein, D. G., J. Biomol. Struct. Dyn.(1993) 11: 685). Similarity, thiosubstitution may lead to structural perturbations in the structure of the duplex. Id. Even in specific protein-nucleic acid contacts, sulfurization of the internucleotide linkages leads to their enhanced binding (Milligan and Uhlenbeck, Biochemistry (1989) 28: 2849; Marshall and Caruthers, Science (1993) 259: 1564). As most of the direct contacts between DNA binding proteins and their binding sites are to the phosphate groups (Otwinowski et al., Nature (1988) 335: 321) the extent of incorporation of modified nucleotides must be controlled for thiosubstituted aptamers to retain target specificity. To this end, the present inventors have developed combinatorial library techniques where the phosphorothioate groups are controllably incorporated during library amplification rather than synthesized into xe2x80x9cpost libraryxe2x80x9d selection oligonucleotide sequences. The method of the present invention provides optimization of the total number of thiophosphates incorporated into the aptamer. Thus, forming novel phosphorothioate oligonucleotides that are nuclease resistant yet have high specificity and high binding is achieved.
In another embodiment of the present invention, a method is provided for thiophosphate selection of nuclease resistant aptamers. In the first step, a random single-stranded combinatorial library is chemically synthesized where at least one set of 5xe2x80x2 and 3xe2x80x2 primer segments flanking a randomized region is included in the sequence. Next, the random combinatorial library is concurrently amplified and modified using enzymatic synthesis, e.g. PCR. The synthetic reaction is provided with a pool of four nucleotides including dA, dT, dG and dC, in which at least one is a thiophosphate.
Although a variety of methods may be employed for isolating aptamers, in one embodiment, target-oligonucleotide complexes are separated from non-binding oligonucleotides by filtration. Oligonucleotides binding to the target molecule are again amplified enzymatically in the presence of the desired thiophosphate nucleotide. The isolation and amplification may be repeated iteratively until at least one oligonucleotide population of defined sequences is obtained.
The method of the present invention was applied to NF-IL6 as a target. A specific binding sequence was obtained using the present invention to obtain a sequence that differs from the known consensus NF-IL6 binding domain. Thus, the present method provides for the generation of novel aptamers that are specific yet nuclease resistant. Although the present NF-IL6 aptamers were generated using monothiophosphate nucleotides, either mono or di-thiophosphate monomers may be used in a PCR reaction or by split synthesis (xe2x80x9cmix and separatexe2x80x9d), to incorporate these backbone modifications into the aptamer. Depending on whether monothiophosphate or dithiophosphate nucleotides are provided, similar or different sequences may be obtained thus expanding the repertoire of target specific aptamers to any given target.
In other aspects, the present invention is direct to the use of PCR to produce chiral duplex phosphothioaptamers using chiral monothiophosphate [xcex1S] nucleotide substrates. PCR amplification is conducted with an achiral or diastereomeric mixture of monothiophosphate [xcex1S] nucleotide substrates. When PCR is conducted using Taq polymerase, e.g., the polymerase selects among available thiophosphate nucleotides and, where an achiral mixture of thiophosphates is provided, the Taq polymerase incorporates only one enantiomer; thus producing a chiral duplex phosphorothioate oligonucleotide. Where for example, the nucleotides are a mix of DATP [xcex1S], dTTP, dGTP and dCTP, PCR amplication of the first single stranded library produces a chiral duplex phosphorothioate at all dA positions other than the primers.
The unique chemical diversity of the present library stems from both the nucleotide base sequence and phosphorothioate backbone sequence. Therefore, the present invention stems from the recognition that polymerases can incorporate chiral phosphorothioates and replicate a random sequence library simultaneously. Indeed this appears to be the first time any backbone modification of either RNA or DNA has been successfully carried out in this in vitro selection technology. The ability of PCR is exploited to amplify small quantities of DNA and enrich these populations for high affinity ligands. Unlike existing technology, however, phosphorothioate aptamers display increased nuclease resistance and are thus better suited for delivery to humans. Furthermore, simply replacing thiophosphates in a selected sequence for normal phosphate ester decreases (or increases) the affinity of the selected ligand for the target at random. Therefore, mere replacement of the ester backbone yields varying results invitro.
The method and identified sequences and compounds are also an improvement over existing antisense or xe2x80x9cdecoyxe2x80x9d oligonucleotides because of their stereochemical purity. Chemically synthesized phosphorothioates are a diastereomeric mixture with 2n stereoisomers with n being the number of nucleotides in the molecule. These preparations are unsuitable for use in humans because only a small fraction of the stereoisomers will have useful activity. Furthermore, the remaining could have potential adverse effects in vivo. In contrast, enzymatically synthesized oligonucleotides are stereochemically pure due to the chirality of polymerase active sites. Inversion of configuration is believed to proceed during incorporation of dNMPxcex1S into the DNA chain.
The present invention provides a method for producing oligonucleotide libraries where the phosphate backbone is substituted with phosphorothioate groups and wherein the substituted sites include at least a portion of the dA sites. The method also includes screening the library for binding to a target molecule or portion thereof that is highly selective, specific and with high affinity.
The present invention also provides oligonucleotide libraries of given or variable nucleotide sequence lengths where at least a portion of the phosphate backbone is substituted with phosphorothioate groups wherein a portion of the dA, dG, dC or dT phosphate sites are substituted with phosphorothioate groups. For example, substantially all of the dA, dG, dC or dT phosphate sites may be substituted with phosphorothioate groups.
The present invention also provides oligonucleotide libraries of given or variable nucleotide sequence lengths where at least a portion of the phosphate backbone is substituted with phosphorothioate groups. A portion of the phosphate sites in the library are substituted with phosphorothioate groups, e.g., where substantially all of phosphate sites are substituted with phosphorothioate groups. Particularly useful are aptamers where phosphate groups in the random nucleotide region of the oligonucleotide sequence library include one or more phosphorothioate backbone bonds.
The present invention contemplates and includes thioated aptamers with multiple substitutions. PCR amplification methods may be used to incorporate up to three dNTPxcex1S""s into DNA. It is anticipated that dNTPxcex1S substitutions will provide the ability to further modulate the nuclease resistance of thioated aptamers and to provide greater diversity to the initial library.
Single-stranded nucleic acids are also known to exhibit unique structures. Single-stranded RNA and DNA may adopt unique structures. The present invention contemplates and includes the selection of single-stranded phosphorothioate aptamers (RNA and DNA). These compounds will find utility in the application of the present methodology and be used compounds that bind to otherwise non-DNA binding proteins (i.e., cell surface receptors, cytokines, etc.).
The present invention includes scaling-up technologies that necessarily follow from the development of methods for high throughput thio-aptamer selection. For example, 96 well microtiter plates may be used to select aptamers to a number of different proteins under varying conditions.
Based on the availability of sequencing using thiophosphate substitution (Nakamaye et al. Nucl Acids Res. (1988) 16: 9947), either backbone may be combinatorially selected and the substitutions identified at any position. The present invention contemplates and encompasses large-scale synthesis of defined stereochemistry and sequence thiophosphate aptamers using stereoregular monothiophosphate oligonucleotides (either R or S configuration at phosphorus for chemical synthesis). Using the present invention, random combinatorial libraries and selection for aptamers with a much greater diversity of structures (7n vs. 4n) are thus possible.
One embodiment of the present invention provides thioselected aptamers specific for the nuclear factor for IL-6 (NF-IL6). NF-IL6 is a basic leucine zipper transcriptional factor involved in the induction of acute-phase responsive and cytokine gene promoters in response IL-6 produces as a result of inflammation. Surprisingly, the thiophosphate selection method provided herein generated a NF-IL6 specific aptamer having a unique sequence different from that obtained using normal backbone selection methods.
Thiophosphate substituted oligonucleotides show reduced nuclease activity and enhanced interaction with proteins in general, not just DNA binding proteins. The present xe2x80x9cthiophosphate-selectionxe2x80x9d method is applicable to the generation of novel aptamers selected against a wide array of targets that may be used therapeutically.
The present aptamers also find utility as biochemical research tools or medical diagnostics agents in cell culture, animal systems, in vitro systems and in the inhibition of high temperature polymerases (hot start PCR). The present invention may be used for the development of new anti-viral, anti-bacterial, anti-cancer, etc. agents. Under both physiological and in vitro conditions the present thioaptamers will provide tools to understand various pathological processes. The aptamers may also be used to develop compounds that have significant anti-pathological activity. Ultimately, they may be delivered as drugs to combat human disease.